Based On The "Gut-kidney Axis" To Explore The Mechanism Of Rhubarb Enema In The Treatment Of CKD By Regulating Intestinal Flora

ObjectivePrevious studies have found that rhubarb enema can slow the progression of CKD,which may be related to regulating the gut microbiota of CKD rats,reducing the levels of IS,LPS and related immune inflammation,and repairing the intestinal mucosal barrier.Due to limited conditions,previous studies could not accurately locate the relevant functional strains and metabolic pathways of rhubarb enema regulating CKD,and whether the intestinal flora was the therapeutic target of rhubarb enema to slow the progression of CKD was still unclear.Therefore,this study was intended to explore the mechanism of rhubarb enema delaying CKD by regulating specific intestinal strains and related metabolic pathways and signaling pathways using the method of multi-omics+bioinformatics analysis.Fecal bacteria transplantation was applied to preliminarily verify the intestinal Microbiota were the target for rhubarb enema to delay CKD.This study was a continuation of the previous study,and will provide an experimental basis for the specific mechanism of rhubarb enema regulating intestinal flora in the treatment of CKD.MethodsThis study uses 5/6 nephrectomy rats as a chronic kidney disease model and is divided into 3 parts:1.Metagenomics was used to detect the specific intestinal strains and their functional genes related to rhubarb enema in the treatment of CKD.After modeling,40 SD rats were randomly divided into model group,model+rhubarb enema group,and another group was sham operation group as control.The sham operation group and model group were given sterile water enema,and the model+rhubarb enema group was given rhubarb enema,once a day for 4 weeks,to detect renal function,urine proteincreatinine ratio,renal pathology,inflammation,intestinal Pathology,intestinal tight junction protein,short-chain fatty acids and other clinical phenotype indicators,metagenomics detection of specific intestinal strains and functional genes of rats in each group,and correlation between clinical phenotypes and differential intestinal strains analyze.2.To investigate the mechanism of rhubarb enema regulating AhR signaling pathway in the treatment of CKD based on metabolomics.Nontargeted metabolomics detection was performed on the serum of each group of rats in part 1 by metabolomics,and the AhR signaling pathway in the intestine and kidney of each group of rats in part 1 was detected by western blot:AhR,CYP1A1,CYP1B1,NF-κB,expression of key proteins.3.Investigating intestinal flora based on fecal bacteria transplantation was the target of rhubarb enema in the treatment of CKD.After modeling,40 SD rats were randomly divided into 4 groups and given corresponding fecal bacteria transplantation.Bacterial transplantation group,fecal bacteria transplantation of rats in sham operation group;model fecal bacteria transplantation group:fecal bacteria transplantation of model group rats;rhubarb fecal bacteria transplantation group:fecal bacteria transplantation of rats in rhubarb group;feces were dissolved in PBS and filtered.The sediment was removed,and the fecal bacteria suspension was transplanted by gavage,once a day,for a total of 4 weeks.After the fecal bacteria transplantation,the renal function,urine protein-creatinine ratio,renal pathology,inflammation,intestinal pathology,intestinal tight junction protein,and other clinical phenotypic indicators of the rats in each group were detected,and serum non-targeted metabolomics analysis after transplantation,16S rDNA were detected of the intestinal flora of rats,and the correlation between each clinical phenotype and the differential intestinal flora was analyzed.Results1.The serum creatinine,blood urea nitrogen,and urine proteincreatinine ratio of rats in the model group were significantly higher than those in the sham operation group(P<0.05).Compared with the model group,the serum creatinine and serum creatinine levels in the model+rhubarb enema group were significantly higher Compared with the model group,there was no significant difference in the ratio of urine protein to creatinine(P>0.05),and there was no significant difference in blood uric acid and blood lipid among the three groups(P>0.05).The results of pathological staining showed that,compared with the sham operation group,the kidneys of the rats in the model group and the model+rhubarb enema group had different degrees of renal tubular brush soft border shedding,atrophy,a large number of mononuclear lymphocytes infiltrated in the tubulointerstitium,and intertubular infiltration.Compared with the model group,the above lesions were lessened in the model+rhubarb enema group.Compared with the sham-operated group,the height of intestinal villi in the model group and the model+rhubarb enema group was lower than that in the sham-operated group,and was accompanied by intestinal mucosal edema,with local mononuclear lymphocyte infiltration.Rhubarb enema can improve the infiltration of intestinal mucosal inflammatory cells and edema.Increased intestinal mucosa villus height.The results of electron microscopy showed that the colon structure in the sham operation group had no obvious abnormality,while in the model group,the microvilli on the surface of epithelial cells were partially absent,cytoplasmic lysis of epithelial cells,cavitation,tight junctions between cells,no desmosomes,and swelling of mitochondrial structures.Rhubarb enema can improve the above lesions.Compared with the sham operation group,the level of short-chain fatty acids in the model group showed a downward trend,but there was no statistical difference(P>0.05),but after rhubarb enema,the level of short-chain fatty acids was significantly higher than that in the model group(P<0.05).Compared with the sham operation group,the levels of inflammatory factors such as IL1β,IL-6,TNF-α,IFN-γ in the model group were significantly increased(P<0.05).Compared with the model group,rhubarb enema could reduce IL1β,TNF-α,IFN-γ and other inflammatory factors.Rhubarb enema can increase lactobacillus-acidophilus,lactobacillus-johnsonil,akkermansiamuciniphila,paraprevotella-xylamiphila,bifidobacterium-animalis and other probiotics,reduce Pathogenic bacteria such as methanobrevibactersmithii,methanosphaera-stadtmanae,escherichia-coil.Further,the correlation analysis of the differential intestinal strains in each group and the phenotypes of renal function,inflammatory factors,intestinal tight junction proteins and short-chain fatty acids was carried out.The results showed that Desulfovibrio-piger and Bacteroides-dorei enriched in the model group were positively correlated with inflammatory factors such as IL-1β,IL-6,TNF-α,and IFN-γ.The relationship was negatively correlated with the intestinal tight junction protein Occludin,and methanobrevibacter-smithii was negatively correlated with short-chain fatty acids.Helicobacter-japonicus,Paraprevotella-xylaniphila,and Lactobacillus-acidophilus enriched in the model+rhubarb enema group were positively correlated with short-chain fatty acids.The functional genes were predicted for the differential strains in each group,and the differential functional genes were obtained.The differential functional genes obtained by the analysis were correlated with phenotypes such as renal function,inflammatory factors,intestinal tight junction proteins and short-chain fatty acids.The results showed that the differentially functional genes of the microbiota in the model group were positively correlated with inflammatory factors such as IL-1β,IL-6,TNF-α,IFN-γ,and negatively correlated with Occludin.The differential functional genes of the model+rhubarb enema group were positively correlated with short-chain fatty acids.2.There were 40 differential metabolic pathways in the sham operation group and the model group,most of which were related to amino acid metabolism,and 2 of them were related to tryptophan metabolism.There were 10 differential metabolic pathways in the model group and the model+rhubarb enema group,one of which is related to tryptophan metabolism.According to the above results,it can be inferred that rhubarb enema may regulate tryptophan metabolism.Therefore,further analysis of the metabolites related to tryptophan metabolism was carried out.It was found that indole,indole-3-carboxyl Acid-o-sulfate,indole-3-lactate,indole-3-acetaldehyde,4 metabolites directly related to tryptophan metabolism,among which indole,indole-3-carboxylic acid-o-Sulfate was significantly increased in model group rats,while indole-3 lactic acid and indole-3 acetaldehyde were increased in model+rhubarb group compared with model group.The key proteins of AhR signaling pathway related to tryptophan metabolism in intestinal and kidney tissues,such as AhR,CYP1A1,and CYP1B1,were further detected.The results showed that the expression of AhR and CYP1B1 proteins in the intestinal tract of the model group was significantly lower than that of the sham operation group(P<0.05).Rhubarb enema can increase the expression of AhR and CYP1B1 proteins in the intestine of CKD rats,especially significantly increase the expression of AhR protein in the intestine of CKD rats(P=0.008).The expression of AhR and CYP1A1 protein in the kidney of the model group was increased compared with that of the sham-operated group,and the protein level of CYP1A1 in the model group was significantly different from that of the sham-operated group(P=0.028).Rhubarb enema decreased the protein of AhR and CYP1B1 in the kidney of CKD rats.The expression level of CYP1A1 in the model+rhubarb group was significantly different from that in the model group(P=0.017).3.After rhubarb enema,the fecal bacteria of the rats in each group were transplanted to CKD model rats,although the four groups of rats after fecal bacteria transplantation had no clinical phenotypes such as renal function,urine protein quantification,and renal and intestinal pathological inflammatory factors.Significant differences,but both in terms of gut microbiota and metabolism.In terms of intestinal flora,the model fecal bacteria transplantation group had the highest abundance of microbiota,the sham-operated fecal bacteria transplantation group had the lowest abundance,and the rhubarb fecal bacteria transplantation group had an abundance between the two groups.The fecal bacterial transplantation of sham-operated rats can increase the abundance of Adlercreutzia,Lactococcus and other genera.Fecal bacterial transplantation in CKD model rats increased:Rikenellaceae,Oscillospira,Sporobacter and other genera abundances.Rhubarb enema increased fecal bacterial transplantation in rats:Butyricicoccus,Prevotellaceae,Peptococcus and other genera abundances.In terms of metabolism,rats in the sham-operated fecal bacteria transplantation group had 26 differential metabolic pathways,such as Cysteine and methionine metabolism,Bile secretion,beta-Alanine metabolism.The rats in the model fecal bacteria transplantation group had 25 differential metabolic pathways including Tryptophan metabolism,Phenylalanine metabolism,and Glutathione metabolism.There were 27 differential metabolic pathways in the rhubarb enema fecal bacteria transplantation group,such as Tyrosine metabolism,Butanoate metabolism,Arginine and proline metabolism etc.Conclusions1.The specific intestinal strains regulated by rhubarb enema were lactobacillus-acidophilus,lactobacillus-johnsonil,akkermansiamuciniphila,paraprevotella-xylamiphila,bifidobact erium-animalis increased,and pathogenic bacteria such as methanosphaera-stadtmanae and escherichia-coil decreased;metabolic pathways:increased short-chain fatty acids,indoleacetaldehyde,indole Indole lactate and other beneficial metabolites,and regulate the intestinal AhR pathway,protect the intestinal barrier,reduce IS and other harmful metabolites,regulate the renal AhR pathway,thereby delaying CKD.2.The intestinal flora may be the target of rhubarb enema treatment for CKD,which needs to be further confirmed.